This grant is focused on providing a detailed mechanism for the role of calcium-activated proteases (calpains) in lens. The overall hypothesis being tested is that proteolysis by calpains is essential for limited truncation, tighter packing of truncated beta-crystallins, and rearrangement of the cytoskeletal elements occurring during normal lens fiber differentiation and maturation. During cataract formation, over-activation of calpains by massive influx of calcium produces rapid proteolysis and light scattering because the truncated crystallins are no longer properly ordered. Studies during the latest grant cycle produced several unexpected findings relevant to this hypothesis: Activity of lens-specific calpain Lp82, a splice variant of muscle p94, and not m-calpain, was most strongly associated with cataract formation in young rodents. Further, calpain 10 was discovered as a third ubiquitous calpain in human lens, and calpain 10 was markedly increased in diabetic cataracts in rats. Lp82, Lp85 (a low-abundance splice variant in lens), and calpain 10 may truncate specific lens proteins or cleave proteins at unique sites necessary for proper lens maturation or pathologic cataract formation. To test these ideas, three specific aims will be pursued: [unreadable] [unreadable] 1. Determine the biochemical properties and substrates of Lp82. [unreadable] [unreadable] 2. Determine if p94 variants assume the role of rodent Lp82 in human lenses and are responsible for unexplained proteolysis in human lenses. [unreadable] [unreadable] 3. Define the roles of calpain 10 in human lens. [unreadable] [unreadable] These studies will provide a rational basis for the use of protease inhibitors to prevent cataracts.